Process for aligning macromolecules by passage of a meniscus and applicaions

ABSTRACT

The subject of the present invention is a process for aligning a macromolecule (macromolecules) on the surface S of a support, characterized in that the triple line S/A/B (meniscus) resulting from the contact between a solvent A and the surface S and a medium B is caused to move on the said surface S, the said macromolecules having a part, especially an end, anchored on the surface S, the other part, especially the other end, being in solution in the solvent A. The subject of the present invention is also a process for detecting, measuring the intramolecular distance of, separating and/or assaying a macromolecule in a sample in which a process of alignment according to the invention is used.

The present invention relates to a method for aligning macromoleculessuch as polymers or macro-molecules with biological activity, especiallyDNA, or proteins. The present invention also relates to the applicationof this method in processes for detecting

for measuring intramolecular distance, for separating and/or forassaying a macromolecule in a sample.

Controlling the conformation of macromolecules represents a majorindustrial challenge, for example in the manufacture of sensors or ofcontrolled molecular assemblies, or alternatively in problems ofdetection and analysis. It may be useful to have an elongated molecularconformation. By way of example, in the case where polymers are graftedon a substrate, it has been proposed to extend them by the action of anelectric field, a flow or with the aid of optical tweezers. Inparticular, in biology, the alignment of DNA—by electrophoresis(Zimmermann and Cox Nucl. Acid Res. 22, p 492, 1994), free flow (Parraand Windle, Nature Genetics, 5, p 17, 1993 and WO 93/22463) or in a gel(Schwartz et al. Science 262, p. 110, 1993 and U.S. Pat. No. 33,531) orwith the aid of optical tweezers (Perkins et al., Science 264 p 819,1994 and also U.S. Pat. No. 5,079,169)—opens numerous possibilities inmapping, or in the detection of pathogens.

These methods only allow in general an imperfect alignment, oralternatively a transient alignment—that is to say that relaxation ofthe molecule occurs once the stress disappears. In the case of opticaltweezers, the method is expensive, is limited to only one molecule at atime, and is difficult to carry out by non-qualified staff.

A special technique for aligning DNA by flow after cell lysis, followedby drying, has been proposed (I. Parra and B. Windle and WO 93/22463).The alignment obtained is very imperfect and nonhomogeneous and numerousnonaligned masses are observed.

The subject of the present invention is a novel and simple method foraligning macromolecules on the surface S of a support, characterized inthat the triple line S/A/B (meniscus) resulting from the contact betweena solvent A and the surface S and a medium B is caused to move on thesaid surface S, the said macromolecules having a part, especially anend, anchored on the surface S, the other part, especially the otherend, being in solution in the solvent A.

It has been observed according to the present invention that the merepassage of a meniscus over molecules of which one part is anchored on asubstrate, the remainder of the molecule existing freely in solutionmakes it possible to align them uniformly, perpendicularly to the movingmeniscus, leaving them adsorbed on the surface behind the meniscus. Thisphenomenon is called “molecular combing” here.

More specifically, the stretching of the free part of the molecule isachieved by the passage of the triple line S/A/B constituting themeniscus between the surface S, the solvent A and a medium B which maybe a gas (in general air) or another solvent.

In a specific embodiment, the meniscus is a water/air meniscus, that isto say that the solvent A is an aqueous solution and the medium B isair.

Furthermore, it is possible to extend the air/water meniscus used herein order to stretch the molecule to other systems such as oil/water orwater/surfactant/air, in particular.

The movement of the meniscus can be achieved by any means of relativemovement of the fluids A and B relative to the surface S. In oneembodiment, the surface S can be removed from the solvent A orconversely, the solvent A can be removed from the surface S.

In particular, the meniscus can be moved by mechanical means, especiallyby pneumatic means by aspirating or blowing a gas, or especially byhydraulic means by pushing or aspirating the solvent A or the medium B.

Thus, the movement of the meniscus can be achieved by gradualevaporation of the solvent A.

When the movement of the meniscus is achieved mechanically, it can beachieved either by translation of the interface A/B, or by translationof the surface S.

In a specific embodiment, the solvent is placed between two supports ofwhich at least one corresponds to the said support of surface S and themeniscus is moved for example by evaporation.

By “support”, there is understood here any substrate whose cohesion issufficient to withstand the passage of the meniscus.

The support may consist, at least at the surface, of an organic orinorganic polymer, a metal especially gold, a metal oxide or sulfide, asemiconductor element or an oxide of a semiconductor element, such assilicon oxide or a combination thereof, such as glass or a ceramic.

There may be mentioned more particularly glass, surface oxidizedsilicon, graphite, mica and molybdenum sulfide.

As “support”, there may be used a single support such as a slide, beads,especially polymer beads, but also any form such as a bar, a fiber or astructured support, and also particles, whether it be powders,especially silica powders, which can moreover be made magnetic,fluorescent or colored as known in the various assay technologies.

The support is advantageously in the form of cover slips. Preferably,the support has little or no fluorescence.

Macromolecules, such as ordinary polymers, or biological polymers suchas DNA, RNA or proteins, can be anchored by ordinary methods on asupport.

The macromolecule to be aligned can be chosen from biologicalmacromolecules such as proteins, especially antibodies, antigens,ligands or their receptors, nucleic acids, DNA, RNA or PNA, lipids,polysaccharides or derivatives thereof.

It was observed according to the present invention, that the stretchingforce acts locally within the immediate vicinity of the meniscus. It isindependent of the length of the molecule, of the number of moleculesanchored, and within a wide range, of the speed of the meniscus. Thesecharacteristics are particularly important for aligning the moleculeshomogeneously and reproducibly.

It is possible, according to the present invention, to add surfactantelements into the solvent A and/or the medium B, which modify theproperties of the interfaces. According to the present invention, thestretching can indeed be controlled by the addition of surfactants, orby an adequate surface treatment.

Too high a surface-macromolecule attraction (for example an excessivelyhigh level of adsorption) can interfere with the alignment of themolecules by the meniscus, these molecules remaining adsorbed at thesurface in a state which is not necessarily stretched. Preferably, thesurface exhibits a low rate of adsorption of the said macromolecule,such that only the anchored molecules will be aligned, the others beingcarried by the meniscus.

However, it is possible to vary the differences in adsorption between apart of the macromolecule, especially its ends, and its other parts (inparticular for long molecules such as DNA or collagen) in order toanchor, by adsorption, the molecules by a part, especially their end(s)alone, the remainder of the molecule existing freely in solution, on awide variety of surfaces and align them by the passage of the meniscusas described above.

The adsorption of a macromolecule onto a surface can be easilycontrolled by means of the pH or of the ionic content of the medium orof an electric voltage applied over the surface. The surface charges andthe electrostatic (repulsive or attractive) interactions between thesurface and the molecule are thus changed, thereby making it possible topass from a state of complete adsorption of the molecule onto thesurface to a total absence of adsorption. Between these two extremecases, there is a range of control parameters where the adsorptionoccurs preferably through the end of the molecules and which willtherefore be used advantageously to anchor them on the surface, and thento align them by the passage of the meniscus.

Once aligned, the molecules adhere strongly to the surface. In the caseof DNA, it was possible to observe them by fluorescence several monthsafter their alignment.

The present invention is therefore very different from the methodproposed by Parra and Windle, because according to the presentinvention, the molecules are anchored on the surface and then uniformlyaligned by the passage of the meniscus, whereas in the Parra and Windlemethod, a hydrodynamic flow is used to stretch the moleculesnonhomogeneously, which molecules will become nonspecifically adsorbedonto the surface.

Other techniques can also result in the stretching and the alignment ofmolecules. Thus, a dynamic orientation of molecules in solution,anchored at one end, can be obtained by electrophoresis or by ahydraulic flow. However, the results observed show that these techniquesare much less efficient than the use of the meniscus.

By “anchoring” of the macromolecule on the surface, there should beunderstood an attachment resulting from a chemical reactivity boththrough a covalent linkage and a noncovalent linkage such as a linkageresulting from physicochemical interactions, such as adsorption, asdescribed above.

This anchorage of the macromolecule can be achieved directly on (orwith) the surface, or indirectly, that is to say via a linkage such asanother molecule, especially another molecule with biological activity.When the anchorage is achieved indirectly, the macromolecule can begrafted chemically on the said linkage, or can interactphysicochemically with the said linkage, in particular when the saidintermediate linkage is a molecule with biological activity recognizingand interacting with the said macromolecule.

In one embodiment, the macromolecule and the said linkage are bothmolecules with biological activity which interact, such as an antigenand an antibody respectively, complementary nucleic acids or lipids. Inthese cases, the noncovalent attachment of the macro-molecule consistsof a linkage of the type: antigen-antibody, ligand-receptor,hybridization between complementary nucleic acid fragments orhydrophobic or hydrophilic interaction between lipids.

Advantage is thus taken of the very high specificity and the very highselectivity of certain biological reactions, especially antigen-antibodyreactions, DNA or RNA hybridization reactions, interprotein reactions oravidin/streptavidin/biotin type reactions, as well as reactions ofligands and their receptors.

Thus, in order to carry out the direct or indirect anchoring of themacromolecule on the surface S, it is possible to use a solid surfacehaving certain specificities. It is in particular possible to usecertain pretreated surfaces which make it possible to attach certainproteins or DNA, whether modified or otherwise.

Such surfaces are commercially available (Covalink, Costar, Estapor,Bangs, Dynal for example) in various forms having at their surface COOH,NH₂ or OH groups for example.

It is, in this case, possible to functionalize the DNA with a reactivegroup, for example an amine, and carry out a reaction with thesesurfaces. However, these methods require specific functionalization ofthe DNA to be attached.

A technique allowing anchorage without prior treatment of the DNA hasalso been described. This process consists in causing a free phosphateat the 5′ end of the DNA molecule to react with a secondary amine of thesurface (NH Covalink surface).

Anchoring by adsorption can be achieved by adsorption of the end of themolecule by controlling the surface charge by means of the pH, the ioniccontent of the medium or the application of an electric voltage over thesurface given the differences in adsorption between the ends of themolecule and its middle part. According to the present invention,nonfunctionalized DNA molecules were thus anchored, by way of example,on surfaces coated with molecules ending with a vinyl or amine groupsuch as polylysine molecules, or various surfaces such as glass, coatedwith silane type molecules ending with vinyl or amine groups oralternatively glass cover slips previously cleaned in an acid bath. Inthis latter case, the surface of the glass indeed has SiOH groups.

In all these cases, the pH range where the DNA is anchored is chosen tobe between a state of complete adsorption and an absence of adsorption,the latter being situated at a more basic pH. It is understood that thistechnique is very general and can be extended by persons skilled in theart to numerous types of surfaces.

It is also possible to functionalize the DNA with a first reactive groupor a protein P₀ in order to cause it to react with a surface coated witha second reactive group or with a protein P₁, which are capable ofreacting specifically with each other respectively, that is to say forexample P₁ with P₀. The P₀/P₁ pair may be a pair of the type:biotin/streptavidin (Zimmermann and Cox) or digoxigenin/antibodydirected against digoxigenin (anti-DIG) for example (Smith et al.,Science 258, 1122 (1992)).

Preferably, the anchoring surfaces will have a low fluorescence level soas not to interfere with the detection of the molecules after theiralignment, in particular if the detection is done by fluorescence.

According to the present invention, a solid support having, under thereaction conditions, a surface having an affinity for only part of themacromolecule, the rest of the macromolecule remaining freely insolution, is preferably used.

In one embodiment, a solid support is used which has at the surface atleast one layer of an organic compound having, outside the layer, anexposed group having an affinity for a type of molecule with biologicalactivity which may be the said molecule itself or a molecule recognizingand/or interacting with it.

The support can therefore have a surface coated with a reactive group orwith a molecule with biological activity.

By “affinity”, there should be understood here both a chemicalreactivity and an adsorption of any type, this under optional conditionsof attachment of the molecules onto the exposed group, modified orotherwise.

In one embodiment, the surface is essentially compact, that is to saythat it limits access by the macromolecule with biological activity tothe inner layers and/or to the support, this in order to minimizenonspecific interactions.

It is also possible to use surfaces coated with a reactive exposed group(for example NH₂, COOH, OH, CHO) or with a macromolecule with biologicalactivity (for example: proteins, such as streptavidin or antibodies,nucleic acids such as oligonucleotides, lipids, poly-saccharides andderivatives thereof) which is capable of attaching an optionallymodified part of the molecule.

Thus, surfaces coated with streptavidin or with an antibody according toknown processes (“Chemistry of Protein Conjugation and Cross-linking”,S. C. Wong, CRC Press (1991)) are capable of attaching a macromoleculehaving, at a specific site, a biotin or an antigen.

Likewise, surfaces treated so as to have single-strandedoligonucleotides can serve in order to anchor on them DNAs/RNAs having acomplementary sequence.

Among the surfaces having an exposed reactive group, there may bementioned those on which the exposed group is a —COOH, —CHO, NH₂, —OHgroup, or a vinyl group containing a double bond —CH═CH₂ which is usedas it is or which can be activated so as to give especially —CHO, —COOH,—NH₂OR OH groups.

The supports with highly specific surfaces according to the presentinvention can be obtained using various processes. There may bementioned by way of example:

-   (A) a layer of carbon-containing, optionally branched, polymer at    least 1 nm thick, having reactive groups as defined below and-   (B) surfaces obtained by depositing or anchoring on a solid support    one or more molecular layers; the latter can be obtained by forming    successive layers attached through noncovalent linkages, as    non-limiting example, Langmuir-Blodgett films, or by molecular self    assembly, this allowing the formation of a layer attached by    covalent linkage.

In the first case, the surface can be obtained by polymerization of atleast one monomer generating at the surface of the polymer the saidexposed group, or alternatively by partial depolymerization of thesurface of a polymer to generate the said exposed group, oralternatively by deposition of polymer.

In this process, the polymer formed has vinyl linkages such as a polyenederivative, especially surfaces of the synthetic rubber type, such aspolybutadiene, polyisoprene or natural rubber.

In the second case, the highly specific surface contains:

-   -   on a support, a substantially monomolecular layer of an organic        compound of elongated structure having at least:        -   an attachment group having an affinity for the support, and        -   an exposed group having no or little affinity for the said            support and the said attachment group under attachment            conditions, but optionally having, after chemical            modification following the attachment, an affinity for one            type of biological molecule.

The attachment can first of all be of the noncovalent type, especiallyof the hydrophilic/hydrohilic and hydrophobic/hydrophobic type, as inLangmuir-Blodgett films (K. B. Blodgett, J. Am. Chem. Soc. 57, 1007(1935).

In this case, the exposed group or the attachment group will be eitherhydrophilic or hydrophobic, especially alkyl or haloalkyl groups such asCH₃, CF₃, CHF₃, CH₂F, the other group being hydrophilic.

The attachment can also be of the covalent type, the attachment groupwill, in this case, react chemically with the support.

Certain surfaces of similar structure have already been mentioned in theelectronic field, especially when the attachments are covalent, L.Netzer and J. Sagiv, J. Am. Chem. Soc. 105, 674 (1983) and U.S. Pat. No.4,539,061.

Among the attachment groups, there must be mentioned more particularlythe groups of the metal alkoxide or semiconductor type, for examplesilane, especially chlorosilane, silanol, methoxy- and ethoxysilane,silazane, as well as phosphate, hydroxyl, hydrazide, hydrazine, amine,amide, diazonium, pyridine, sulfate, sulfonic, carboxylic, boronic,halogen, acid halide, aldehyde groups.

Most particularly, as attachment group, groups capable of cross-reactingwith an adjacent equivalent group, to give cross-linkages will bepreferably used; for example they will be derivatives of the metalalkoxide or semiconductor type, for example silane, especiallydichlorosilane, trichlorosilane, dimethoxysilane or diethoxysilane andtrimethoxy- or triethoxysilane.

The choice of the attachment group will obviously depend on the natureof the support; the silane-type groups are quite suitable for covalentattachment on glass and silica.

As regards the exposed groups, irrespective of the surface, they will bepreferably chosen from ethylenic groups, acetylenic groups or aromaticradicals, primary, tertiary or secondary amines, esters, nitriles,aldehydes, halogens. But they may be most particularly the vinyl group;indeed, the latter can be either chemically modified after attachment togive, for example, a carboxylic group or derivatives of carboxylicgroups such as alcohol groups, aldehyde groups, ketone groups, acidicgroups, primary, secondary or tertiary amines, or to lead to apH-dependent direct anchoring of the biological macromolecules such asnucleic acids and proteins, without chemical modification of the surfaceor of the macromolecules.

Preferably, the chains connecting the exposed group to the attachmentgroup are chains carrying at least 1 carbon atom, preferably more than 6and in general from 3 to 30 carbon atoms.

As regards the support itself, the use of glass, surface-oxidizedsilicon, a polymer or gold with or without pretreatment of the surface,is generally preferred.

In the case of glass or silica, there can be used advantageously theknown techniques for surface functionalization using silane derivatives,for example: Si—OH+Cl₃—Si—R—CH═CH₂ gives Si—O—Si—R—CH═CH₂, R consistingfor example of (CH₂)₄. Such a reaction is known in literature, with theuse of ultrapure solvents. The reaction leads to a layer of moleculeshaving their C═C end at the surface exposed to the outside.

In the case of gold, this being optionally in the form of a thin layeron a substrate, the known techniques for surface functionalization usethiol derivatives, for example: Au+HS—R—CH═CH₂ gives Au—S—R—CH═CH₂, Rconsisting for example of (CH₂)₄. Such a reaction is described in liquidmedium and leads, like the preceding trichlorosilane-silica reaction, toa layer of molecules having their C═C end at the surface exposed to theoutside.

Of course the term “support” encompasses both a single surface such as aslide, but also particles, either silica powder or polymer beads, andalso ordinary forms such as a bar, a fiber or a structured support,which can moreover be made magnetic, fluorescent or colored, as is knownin various assay technologies.

Preferably, the support will be chosen so as to have no or littlefluorescence when the detection will be carried out by fluorescence.

The surfaces obtained according to methods (A) or (B) above have:

-   (i) a very low level of intrinsic fluorescence, when necessary, a    fluorescence background noise (with a typical surface area of    100×100 μm) smaller than the fluorescence signal of a single    molecule to be detected;-   (ii) the possibility of detecting isolated molecules with an S/N    ratio independent of the number of molecules, which is possible by    virtue of various techniques with a high S/N ratio which are    described below and which are based on the identification of the    presence of a macroscopic marker having a weak nonspecific    interaction with the surface.

The surfaces thus obtained are preferably coated with a macromoleculewith biological activity chosen from:

-   -   proteins,    -   nucleic acids    -   lipids    -   polysaccharides and derivatives thereof.

Among the proteins, there should be mentioned antigens and antibodies,ligands, receptors, but also products of the avidin or streptavidintype, as well as derivatives of these compounds.

Among the RNAs and DNAs, there should also be mentioned the α, βderivatives as well as the thio derivatives and mixed compounds such asPNAs.

It is also possible to attach mixed compounds such as glycopeptides andlipopolysaccharides for example, or alternatively other elements such asviruses, cells in particular, or chemical compounds such as biotin.

The attachment of the biological macromolecules may be covalent ornoncovalent, for example by adsorption, hydrogen bonds, hydrophobic,ionic interactions for example, in which case cross-linking can beadvantageously carried out in the molecules grafted by known methods(“Chemistry of Protein Conjugation and Crosslinking”, S. C. Wong, CRCPress (1991)) and this in order to enhance their cohesion.

As mentioned above, it is possible to have an exposed group which allowsdirect reaction with molecules with biological activity, but it is alsopossible to envisage that the exposed group is treated, afterattachment, so as to be converted, as indicated above, to a hydroxyl,amine, alcohol, aldehyde, ketone, COOH radical or a derivative of thesegroups before attachment of the biological molecule.

When such groups were exposed, techniques for attachment of proteinsand/or of DNA for example are known, they are indeed reactionsimplemented for surfaces which are already used for biological analysis,especially for Costar surfaces, Nunc surfaces or microbeads such asEstapor, Bang and Dynal for example, on which molecules of biologicalinterest, DNA, RNA, PNA, proteins or antibodies for example, areanchored.

In the case where the exposed group is a —CH═CH₂ radical which is calledhereinafter “surface C═C” or “surface with ethylenic bond”, no documentexists which mentions direct anchoring, in particular of DNA or ofproteins.

Within the framework of the present invention, it has been demonstratedthat these surfaces have a highly pH-dependent reactivity. Thischaracteristic makes it possible to anchor the nucleic acids or theproteins using pH regions and often with a reaction rate which can becontrolled by the pH.

The anchoring of DNA can be carried out by its end onto a surface havinggroups with ethylenic double bonds, by bringing the DNA into contactwith the surface at a pH of less than 8.

In particular, the reaction is carried out at a pH of between 5 and 6,and is then stopped at pH 8.

Thus, for DNA at pH 5.5, the anchoring reaction is complete in one hour(if it is not limited by diffusion) and occurs via the ends. At pH 8 onthe other hand, the attachment is very low (reaction rate of 5 to 6orders of magnitude smaller). This pH dependent attachment effectspecific for the ends, is an improvement compared with the othersurfaces which require functionalization of the DNA (biotin, DIG, NHS,and the like) or specific reagents (carbodiimide, dimethyl pimelidate)which form a peptide or phosphorimide linkage between —NH₂ and —COOH or—POOH.

It is also possible to carry out the anchoring of DNA by adsorption ofits ends alone onto a surface coated with polylysine or a silane groupending with an amine group.

In order to carry out the anchoring of the DNA by its end on a surfacecoated with an amine group, the DNA is brought into contact with thesurface at a pH of between 8 and 10.

Likewise, it is possible to carry out the anchoring of DNA by its endonto a glass surface treated beforehand in an acid bath, by bringing theDNA into contact with the said surface at a pH of between 5 and 8.

It goes without saying that the present invention involves, in the samespirit, the optionally pH-dependent attachment of all macromolecules ofbiological interest.

Likewise, these surfaces can anchor proteins directly (protein A,anti-DIG, antibodies, streptavidin and the like). It has been observedthat (i) the activity of the molecule can be preserved and (ii) that thereactivity of the prepared surface (initially C═C) is completelyovershadowed in favor of the sole reactivity of the molecule ofinterest. It is therefore possible, starting with a relatively highinitial reactivity, to pass to a surface having a very highly specificreactivity, for example that of specific sites on a protein.

By anchoring a specific antibody on the surface (for example anti-DIG),a surface is created whose reactivity is limited to the antigen (forexample the DIG group). This indicates that the initial chemical groupsare all occulted by the antibodies grafted.

It is also possible to graft onto the reactive (chemically orbiochemically) surfaces other molecules with biological activity,especially viruses or other components: membranes, membrane receptors,polysaccharides, PNA, in particular.

It is also possible to attach the product of a reaction of biologicalinterest (for example PCR) onto the prepared surfaces.

The process according to the present invention allows the detectionand/or the quantification of biological molecules, but also themeasurement of intramolecular distance, the separation of certainbiological molecules, especially a sample using antigen/antibody and/orDNA/RNA coupling techniques.

In particular, the subject of the present invention is a process fordetecting a macromolecule, consisting of a DNA sequence or a protein ina sample, according to the present invention, characterized in that:

-   -   the sample corresponding to solvent A, in which the said        macromolecule is in solution, is brought into contact with the        surface of the support under conditions for forming a DNA/DNA,        DNA/RNA hybrid or for forming the protein/protein reaction        product,    -   the hybrid or a macromolecule for labeling the hybrid or the        reaction product being anchored in one part, the remainder being        free in solution, it is stretched by the movement of the        meniscus created by the contact between the solvent and the        surface in order to orientate the hybrids or the said labeling        macromolecules and the measurement or the observation of the        hybrids or of the said labeling macromolecules thus orientated        is carried out.

Advantageously, the attached DNA and the DNA of the sample are coloreddifferently and after stretching, the position of the complementarysequence relative to the end of the sample DNA is measured.

Appropriately, the ELISA or FISH detection methods can be used.

The DNA sample may be the product or the substrate of a DNA enzymaticamplification such as PCR, that is to say that the amplification of theDNA can be carried out once it has been anchored and aligned accordingto the process of the invention or before its anchoring or itsalignment.

The passage of the meniscus, by stretching the molecules linearly, inthe form of rods, renders them more easily detectable if they arelabeled. Moreover, these elongated molecules are stable to the open airand can be observed even after several months, without showing apparentdegradation.

During a rehydration, the DNA molecules can remain adsorbed andelongated. Furthermore, it is possible to carry out a hybridization onthe elongated molecule.

Furthermore, exhibiting a signal which is correlated and of uniformorientation by virtue of their stretching, these molecules are distinctfrom the surrounding noise. It is therefore easy to ignore the dusts,the inhomogeneities, which have no special spatial correlation. Thealignment is also important because in solution, the molecules in theform of a random cole fluctuate thermally, thereby causing very highvariations in their fluorescence signal gathered preferably with a smalldepth of field and limits their observation. The present inventiontherefore allows the observation of isolated molecules with a very highsignal to noise (S/N) ratio.

It is remarkable that this ratio is independent of the number ofmolecules anchored. The S/N ratio posed by the detection of a moleculeis the same as that for 10,000. Furthermore, this stretching techniquemakes it possible to easily discriminate between molecules of varyinglengths.

It is advantageously possible to proceed to the follow ing stages inorder to further improve the S/N ratio:

-   -   The molecule being stationary, its fluorescence signal can be        integrated.    -   Microscopic observation presents a reduced field (typically 100        μm×100 μm with a ×100 immersion lens, N.A.=1.25). For a 1 cm²        sample, scanning can be carried out, or it is possible to        envisage the use of lower magnification lenses (×10 or ×20) but        with a high numerical aperture.    -   The rods being always parallel, it is possible to envisage an        optical spatial filtration method in order further to increase        the S/N ratio.    -   Other global fluorescence methods can be envisaged such as those        described in European Patent Application EP 103426.    -   The linearization of the molecules is observed both within the        framework of a physicochemical anchoring and in the case of        immunological type linkages (DIG/anti-DIG).    -   Once the surface is in the open air, the DNA molecules are        stable (they maintain their integrity even after several weeks)        and fluorescent. This property can be advantageously used in        order to defer the anchoring stage and the locating/counting        stage for the molecules anchored, if this detection is done for        example, but without being limited thereto, by fluorescence        microscopy. Such a use is covered by the present invention.    -   A double (or multi) fluorescence technique can possibly be used        to improve the S/N ratio or to detect a double functionality.

The stretched molecules can be revealed by various enzymological methodson others, such as fluorescence, or the use of radioactive ornonradioactive probes. Their detection can be achieved by measuring aglobal signal (for example the fluorescence) or by individualobservation of the molecules by optical fluorescence microscopy,electron microscopy, local probe methods (STM, AFM and the like).

Thus in general, the present invention allows the detection, separationand/or assay of a molecule in a sample, by a process characterized inthat a surface capable of specifically attaching the said molecule isused, and in that the detection, separation or assay are carried outusing a reagent, fluorescent or otherwise, which detects the presence ofthe attached molecule.

Among the reagents, there are fluorescent reagents and nonfluorescentreagents.

The fluorescent reagents contain fluorescent molecules, advantageouslychosen to be long molecules of size greater than 0.1 μm and reactingspecifically, directly or indirectly, with the pretreated surfaces. Forexample, but with no limitation being implied, a double-stranded DNAmolecule stained by means of fluorescent probes (ethidium bromide, YOYO,fluorescent nucleotides and the like) capable of anchoring directly viaone or more ends on a surface optionally having a vinyl or amine typegroup and the like, especially by a judicious choice of the pH or of theionic content of the medium or by application of an electric voltageover the surface.

It is also possible to use a special functionalization of the molecule(DIG, biotin and the like) in order to anchor it at one or more pointson a surface having complementary sites (anti-DIG, streptavidin and thelike).

Nonfluorescent reagents allowing the detection of molecules previouslyaligned according to the present invention may consist especially ofbeads or micro-particles anchored via another molecule attachedspecifically, directly or indirectly, to the aligned molecule and havingonly a weak nonspecific interaction with the surface.

For example, there may be mentioned Dynal beads coated with streptavidinpermitting anchoring on biotinylated DNA aligned according to thepresent invention.

Depending on whether the desired molecule is detected directly byfluorescence or indirectly by means of the above reagents, the detectionwill be described as “direct detection” or “flag detection”.

In order to limit the problems associated with too slow reaction times,the diffusion times of the reagents towards the surface can beadvantageously reduced using small reaction volumes. For example, butwith no limitation being implied, by carrying out the reaction in avolume of a few microliters determined by the space between two surfacesof which one is treated so as to have reactive sites and the other isinert or treated so as not to have reactive sites, under the reactionconditions

The detection of the number of aligned molecules can be carried out on asmall number of molecules (typically 1 to 1000), by a low-noisemacroscopic physical test requiring neither electron microscope norradio-activity nor necessarily PCR.

The alignment and detection processes according to the present inventionare capable of being carried out by persons having only limitedlaboratory experience.

The specificity of certain biological reactions may be limited. Thus,within the framework of the hybridization, the hybrids may be imperfect(reactions with other sites) while having a reduced number of pairingand therefore a lower quality of binding. The present invention alsocovers the possible use of a stage for testing the quality of the bondsobtained. This test makes it possible to dissociate the products weaklyand nonspecifically paired by adsorption, hydrophobic forces, imperfecthydrogen bonds, imperfect hybridization, in particular.

Accordingly, the invention also relates, in a detection or assay processas described above, to a process where the product of the reactionbetween the molecule with biological activity and the sample molecule issubjected to a stress in order to destroy the mismatches before thedetection.

This process offers, in addition to the possibility of destroying themismatched pairs, the possibility of orientating the products of thecoupling, which facilitates the measurements or the observations.

It is thus possible to apply to the surfaces, after attachment of thecomplementary elements, a stress which may consist of the single orcombined use of:

-   -   centrifugation,    -   gradient of magnetic field applied to the non-fluorescent        reagents taken, in this case, to include magnetizable or        magnetic microbeads,    -   stirring,    -   liquid flow,    -   meniscus passage,    -   electrophoresis    -   temperature variation, and/or temperature gradient.

The number of systems to have remained intact or to have been destroyedis then determined by the low-noise detection techniques describedabove.

The alignment and detection techniques described according to thepresent invention can be used for numerous applications among which, butwith no limitation being implied:

-   -   the identification of one or more elements of DNA or RNA        sequence which can be advantageously used for the diagnosis of        pathogens or the physical map of a genome. In particular, the %        techniques described above make it possible to obtain a physical        map directly on genomic DNA without the intermediate use of a        cloning stage. It is understood that the combed molecule having        been stretched relative to its crystallographic lengths,        relative measurements are carried out. It is thus possible to        measure the size of the DNA fragments and the distance between        fragments, with a resolution of the order of 200 nm by optical        methods or of the order of 1 nm by the use of near field methods        such as AFM or STM in order to visualize and measure the        distance between probes on the aligned DNA.        This naturally leads to:    -   1) the detection of deletions, additions or translocations of        genomic sequences, in particular in the diagnosis of genetic        diseases (for example Duchesne's myopathy);    -   2) the identification of promoters of various genes by measuring        the distance between the regulatory sequences and those        expressed;    -   3) the localization of regulatory proteins by identifying their        position along the DNA or the position of their target sequence;    -   4) the partial or complete sequencing by measuring the distance        using near field microscopy (for example AFM or STM) between        hybridized probes belonging to a base oligonucleotide of given        length;    -   the enzymatic amplification in situ on aligned DNAs;    -   the improvement of the sensitivity of ELISA techniques with the        possibility of detecting a small number (possibly less        than 1000) of immunological reactions.

Thus, physical mapping can be carried out directly on a genomic DNAwithout the intermediate use of a cloning stage. The genomic DNA isextracted, purified, optionally cleaved with one or more restrictionenzymes and then combed on surfaces according to the process of thepresent invention.

The position and size of the desired gene on the genomic DNA are thendetermined by hybridization with probes specific for the said gene,especially extracted from parts of the complementary DNA (cDNA) of theproduct of the said desired gene.

Similarly, by hybridizing a genomic DNA combed, then denatured withtotal cDNA labeled by fluorescence or any other marker allowing thehybrid to be localized, the position, size and number of exons of thegene in question are identified and its size and its geneticorganization (exons, introns, regulatory sequences) are deducedtherefrom.

The position of the gene having been determined as described above orbeing known, it is then possible to identify, by hybridization, theflanking sequences of the gene. For that, the procedure isadvantageously carried out by hybridization with labeled probes,obtained for example from an oligonucloetide library, in order toidentify two or more probes which hybridize on either side of the gene.

From this determination, it is then possible, by enzymatic amplificationtechniques, for example in situ PCR (Nuovo G. J. PCR in situhybridization: protocols and applications, Raven Press (1992)) toamplify the fragment delimited by the flanking probes which can serve asprimers for the reaction, which fragment may contain the desired genewith its regulatory regions which may be tissue- or development-specificand which can then be isolated and purified.

The procedure can also be carried out by in situ polymerization onprimers extracted from the cDNA of the gene in question in order toextract DNA fragments complementary to the flanking regions of the geneas mentioned by Mortimer et al. (Yeast 5, 321, 1989). These fragmentscan then serve in the preparation of primers for a process of enzymaticamplification of the gene and of its flanking sequences.

The methods cited by A. Thierry and B. Dujon (Nucl. Acid Research 205625 (1992)) for inserting, by homologous recombination or randomly,known endonuclease-specific sites into a genomic DNA or a genomic DNAfragment, may also be used. The combing of this DNA allows theidentification of the gene of interest and of the specific sitesinserted, by the in situ hybridization methods described above. Fromthis identification and preferably, if the sites of interest are regionsof interest which are close to the gene, they will be used as primer fora reaction of enzymatic amplification (in situ and the like) of the genein question and of its flanking sequences.

The amplification of the desired gene then proceeds using knownenzymatic amplification techniques such as PCR on the amplified fragmentas described above, using primers which can be reached by the exonsconstituting the cDNA, or primers corresponding to flanking sequences.

By the combing of genomic DNA and the like, it is also possible todetermine, by hybridization, the presence or the absence of regulatorysequence of a specific proximal gene, from which the possible familiesof proteins for regulating this gene (for example: helix-loop-helix,zinc-finger, leucine-zipper) will be determined.

The specific reactions between particular DNA/RNA/PNA sequences andanother molecule (DNA, RNA, protein) can occur before or after aligningthe molecules according to the prevent invention.

Thus, in the field of genetic diagnosis and physical mapping, the knownmethods of FISH (Pinkel et al., Proc. Nat. Acad. Sci. USA 83, 2934(1986)) are advantageously used to hybridize single-strandedoligonucleotides labeled with DNA first aligned, and then denatured. Therevealing of the hybrids will be carried out using known techniques(fluorescence, microbeads and the like) with a resolution in themeavurement of the distances ranging from 0.2 μm (optically) to 1 nm (bynear field microscopy; AFM, STM and the like).

Alternatively, it is possible to first hybridize fluorescently labelledDNAs to single-stranded DNA in solution, and then to align thisconstruct by action of the meniscus after having converted it todouble-stranded DNA and anchored it on an appropriate surface.

It is also possible to use the present invention for detecting theprevence of a pathogen. By way of example, the procedure can be carriedout in two different ways depending on whether the recognition reaction(hybridization, attachment of proteins) occurs before or after alignnentby the meniscus.

Thus, by way of example one or several oligonucleotide probes areanchored in one or more regions of a surface. The hybridization of thepotentially pathogenic DNA is carried out in situ under stringentconditions so as to anchor only the hybridized molecules. Theirdetection and quantification is carried out after alignnent by themeniscus according to the present invention.

Alternatively, the potentially pathogenic DNA is first aligned, thendenatured and hybridized with an oligo-nucleotide probe under stringentconditions. The detection of the hybrid is then carried out by knownmethods, especially by the FISH method, as described above.

Similarly, it is possible to detect the presence (or the absence) of asmall numher of molecules, such as proteins, lipids, sugars or antigens.A minor modification of the ELISA techniques will be advantageouslycarried out, the usual detection method being replaced by the detectionof a fluorescent molecule aligned according to the present invention andcoupled to one of the reagents of the ELISA reaction.

Moreover, as mentioned by R. R. Allan et al. (U.S. Pat. No. 84,114),genetic mapping can be carried out by measuring the size of the DNAfragments. Now, the novel techniques for stretching molecules describedabove (stretching by meniscus) allows the length of the stretchedmolecules to be measured, and this on a very small sample (a fewthousandths of molecules).

It is for example possible, but with no limitation being implied, tocarry out the procedure in the following manner:

A DNA sample is fragnented (by means of restriction enzymes) stainedwith a fluorophore and then anchored on a surface. The molecules arethen stretched by the meniscus and the size of the stretched fragmentsis determined by optical fluorescence microscopy with a resolution and amaximum size of the order of 1000 bp (0.3 μm).

For this purpose, but also if it is desired to align very long molecules(≧10 μm), known techniques will be advantageously used in order to limitthe degradation of long macromolecules during their handling (byhydrodynamic shearing).

Thus, as mentioned by D. C. Schwartz, condensation of the molecules willbe advantageously carried out by means of a condensing agent (forexample spermine or an alcohol) during their handling. Optionally, theirdecondensing will occur during contact between the solvent A and theanchoring surface S.

In order to reduce the degradation of the macro-molecules duringstretching by the meniscus, meniscus translation techniques will be usedwhich minimize hydrodynamic shearing. For example, but with nolimitation being implied, by very slowly removing (≦200 μ/sec) thesurface S from a substantial volume (≧100 μl) of the solvent A.

The subject of the present invention is also a surface having one ormore types of aligned macro-molecules obtained according to the presentinvention. In particular, it is possible to obtain a surface or a stackof surfaces having anisotropic optical or electrical properties.

The subject of the present invention is also a process for aligning anddetecting DNA in which the DNA is stretched by an aligning processaccording to the invention, then denatured and hybridized with specificprobes in order to determine the position or the size of one or morespecific sequences.

The subject of the present invention is also a process for the physicalmapping of a gene on a genomic DNA in which the DNA is aligned ordetected according to a process of the invention.

In particular, the position and the size of the desired gene on thegenomic DNA are determined by hybridization with probes specific for thesaid gene to be mapped.

A subject of the present invention is also

-   -   a kit useful for carrying out a mapping process according to the        invention, consisting of total genomic DNA from a reference        host,    -   a support having a surface permitting the anchoring and the        alignment of the patient's DNA in accordance with the process of        the invention    -   probes specific for the gene(s) to be mapped and reagents for        the hybridization and the detection of the DNA.

The subject of the present invention is also a process for aligning anddetecting DNA in which the DNA is stretched, then denatured andhybridized with specific probes in order to determine the prevence orthe absence of one or more DNA sequences in the said aligned DNA.

The prevent invention allows the implementation of a process for thediagnosis of a pathology related to the presence or the absence of a DNAsequence specific for the pathology in which an alignment processaccording to the invention is used.

The subject of the prevent invention is also a kit useful for carryingout a diagnostic process according to the invention, characterized inthat it contains a support whose surface permits the anchoring and thealignsent of the patient's DNA according to a process of the invention,probes specific for the gene involved in the sought pathology andreagents for the hybridization and the detection of the DNA.

The subject of the prevent invention is also a kit useful for carryingout a diagnostic process according to the invention, characterized inthat it contains a support whose surface has probes specific for thegene involved in a pathology, in particular optionally labeledpathogenic DNA, which are aligned according to the process of thepresent invention and optionally denatured; the reagents for preparingand labeling the patient's DNA for its hybridization (for examplephotobiotin, nick translation or random priming kit) and reagents forthe hybridization and the detection of the DNA according to the in situhybridization techniques as described above.

It is understood that combed probes relating to different pathogens maybe prevent on different supports or on the same support. Theidentification of the corresponding pathogen can be carried out afterhybridization, either spatially (the different probes are spatiallyseparated for example by photochemical anchoring before their combing)or by a difference in the fluorescence spectrum of the differenthybrids, resulting from a prior differential labeling of the probes.

Finally, the subject of the present invention is a process for preparinga gene in which the position of the said gene on the genomic DNA alignedby the process according to the invention is identified by means of aprobe specific for the said gene, the sequence of the said gene andoptionally its flanking sequences are amplified by enzymaticamplification, in particular by in situ PCR.

The present invention therefore makes it possible to carry out a processfor replacing a gene in the genome of an eukaryotic cell by targetedinsertion of a foreign gene by means of a vector containing the saidforeign gene prepared according to the above gene preparation process.

The targeted insertion can be carried out according to the techniquesdescribed in WO90/11354 by transfecting eukaryotic cells with a vectorcontaining the said foreign DNA to be inserted flanked by two genomicsequences which are contiguous to the devired site of insertion in therecipient gene. The insert DNA may contain either a coding sequence, ora regulatory sequence. The flanking sequences are chosen so as to allow,by homologous recombination, depending on the case, either theexpression of the coding sequence of the insert DNA under the control ofthe regulatory sequences of the recipient gene, or the expression of acoding sequence of the recipient gene under the control of a regulatorysequence of the insert DNA.

The genomic genes and the cDNAs obtained using the process forlocalizing genes according to the invention can be inserted intoexpression vectors capable of being inserted into a prokaryotic,eukaryotic or viral host cell. The derived proteins, polypeptides andpeptides are included in the present invention.

The following description is made with reference to the accompanyingfigures in which:

FIG. 1 schematically represents the detection of a pathogen in afluorescent DNA molecule by hybridization with an anchor molecule;

FIG. 2 schematically represents genetic mapping by extension of DNA andthe use of a marker DNA;

FIG. 3 schematically represents the detection of an immunologicalreaction (ELISA) by means of a “flag” molecule: a fluorescent DNA-usedas reaction marker;

FIG. 4 is a fluorescence micrograph showing the extension of λ phage DNAby the progression of the meniscus, DNA molecules in solution stretchedby the evaporation flow parallel to the meniscus can be seen on theleft, DNA molecules in the open air after being stretchedperpendicularly to the meniscus can be seen on the right;

FIGS. 5(a) and 5(b) are fluorescence micrographs showing, respectively,a DNA labeled with digogixenine [sic] (DIG) on a surface coated withanti-DIG and stretched by the meniscus, and, as control, an unlabeledDNA on an anti-DIG surface; the very high specificity of the surfacesand the absence of nonspecific anchoring will be noted;

FIG. 6 represents the schematic-representation of the spread of the DNAby passage of the meniscus. The DNA in solution is anchored on a treatedsurface. The DNA solution is covered with an untreated round cover slip;

FIG. 7 represents histograms of the length of the combed λ DNA moleculeson glass surfaces:

-   -   a) coated with silane molecules ending with an amine group,    -   b) coated with polylysine,    -   c) cleaned in a hydrogen peroxide/sulfuric acid mixture;

FIG. 8 represents combed DNA molecules on glass surfaces coated withpolylysine. It can be noted that the molecules attached by their twoends form loops;

FIG. 9 represents YACs combed by removal of a treated cover slip in asolution of these molecules;

FIG. 10 shows the identification of the presence and the size of acosmid on a YAC by in situ hybridization.

In the “diagnostic mode, the probes (the “anchors”) possess a reactivegroup (DIG, biotin and the like) capable of anchoring specifically on asurface according to the present invention (having for example asanchoring site an anti-DIG antibody or streptavidin). The detection ofthe anchoring reaction can be carried out directly by detection of thefluorescence of the DNA molecule stained by fluorescent molecules(ethidium bromide, YOYO, fluorescent nucleotides) (FIG. 1). It can alsobe carried out indirectly by detection of a flag molecule”: a reagentcapable of attaching to the DNA/RNA molecule (for example byhybridization, protein-DNA interaction and the like), but having noaffinity for the anchoring sites of the probe.

In the “mapping” mode, in situ hybridization techniques (FISH) can beused. It is also possible to envisage other techniques, for example byhybridizing in solution DNA with probes having fluorescent reagentsaccording to the present invention. The detection of the position of theprobes is carried out after aligning the molecule according to theprevent invention.

EXAMPLE 1

Materials and Methods

The λ DNA and the monoclonal antibody (anti-DIG) are obtained fromBoehringer-Mannheim. The trichlorosilanes are obtained fromRoth-Sochiel. The fluorescent nucleic probes (YOYO1, YOYO3 and POPO1)are obtained from Molecular Probes. The ultraclean glass cover slips areobtained from Erie Scientific ((ESCO) cover slips). The magneticparticles are obtained from Dynal. The microscope is a Diaphot invertedmicroscope from NIKKON, equipped with a Xenon lamp for epifluorescenceand a Hamamatsu intensified CCD camera for the visualization.

Surface Treatment

Glass cover slips are cleaned for one hour by UV irradiation under anoxygen atmosphere (by formation of ozone). They are then immediatelyplaced in a desiscator previously purged of traces of water by an argonstream. A volume of about 100 to 500 μl of the appropriatetrichlorosilane (H₂C═CH—(CH₂)_(N)—SiCl₃) is introduced into thedesiccator, from which the surfaces are removed after about 12 hours(n=6) or 1 hour (n=1). Upon taking out, the surfaces are clean andnonwetting.

The functional groups of these double bond surfaces (H₂C═CH—) can beconverted to carboxyl groups (—COOH) by soaking the treated cover slips,as described above, for about ten minutes in a solution of 25 mg KMnO₄,750 mg NaIO₄ in 1 l of water, then by rinsing them three times inultrapure water.

The cover slips thus functionalized can react with proteins. A volume of300 μl of an aqueous solution (20 μg/ml) of proteins (protein A,streptavidin and the like) is deposited on a cover slip functionalizedwith a (H₂C═CH—) group. This cover slip is incubated for about two hoursat room temperature, then rinsed three times in ultrapure water. Thesurfaces thus treated are clean and wetting. The surfaces treated withprotein A can then react with an antibody, for example an anti-DIGantibody, by incubating in a solution of 20 μg/ml of antibody.

Moreover, on the surfaces having carboxyl groups, it is possible tograft oligonucleotides having an amine end (—NH₂), 200 μl of a solutionof MES (50 mM, pH 55), carbodiimide (1 mg/ml) and 5 μl ofamino-oligo-nucleotide (10 pmol/140 μl) are deposited on a carboxylatedsurface and incubated for about 8 hours at room temperature. The coverslip is finally rinsed three times in NaOH (0.4 M) and then four timesin ultrapure water. The cover slips thus prepared can hybridize DNAscomplementary to the anchored oligonucleotide.

Anchorina of Native DNA on a Double Bond Surface

A drop of 2 μl of a fluorescence-labeled λ DNA (YOYO1, POPO1 or YOYO3,but withno specific end labelling) of varying concentration and indifferent buffers (total number of molecules<10⁷) is deposited on apretreated cover slip (C═C) and covered with an untreated glass coverslip (diameter 18 mm). The preparation is incubated for about 1 hour atroom temperature in an atmosphere saturated with water vapor. In a 0.05M MES buffer (pH=5.5), a virtually general anchoring of the DNAmolecules is observed. In contrast, in a 0.01 M Tris buffer (pH=8),there is practically no anchored molecule (ratio>10⁶). This dependencecan make it possible to control the activation/deactivation of surfaces(with respect to DNA) via the pH.

The action of the meniscus on the molecule is limited to its immediatevicinity. The part of the molecule in solution in front of the meniscusfluctuates freely and the part left stuck on the surface behind themeniscus is insensitive to a change in the direction of the meniscus.The extension rate of the molecule is therefore uniform and independentof its size.

Alignment and Detection of the Anchoring by the Action of the Meniscus

By transferring the preceding preparation to a dry atmosphere, thesolution, upon evaporating, will stretch the DNA molecules anchored onthe surface, perpendicularly to the meniscus. The capillary force on theDNA molecule (a few tens of picoNewtons) is indeed sufficient tocompletely stretch the molecule (greater than the entropic elasticityforces), but too weak to break the bond between the end of the moleculeand the treated surface. The DNA having been fluorescence labeled, thestretched molecules (total length about 22 μm) can be individually andeasily observed. The anchoring between the surface and the DNA beinglimited to the ends, it is possible to stretch either DNA of λ phage, ofYAC or of E. coli (total length greater than 400 μm). This DNApreparation, stretched, fluorescent and in the open air, is stable forseveral days and can be observed in a nondestructive manner, byepifluorescence (Nikkon Diaphot inverted microscope with a ×100 lens,O.N.: 1.25).

Specific Anchoring and Detection

By treating the surfaces as described above with a specific monoclonalantibody, it is possible to control their specificity very precisely.Thus, the specificity of anti-DIG treated surfaces was tested inrelation to λ DNA hybridized with an oligonucleotide complementary toone of the Cos ends and possessing a digoxigenin group (DIG) and inrelation to nonhybridized DNA. In the first case, a virtually generalextension of the anchored molecules, by the action of the meniscus, wasobserved. In the second case, only a few anchored DNA molecules (<10)were observed in the whole sample. It is therefore estimated that thespecificity of the method according to the invention is greater than10⁶.

λ DNAs were also hybridized with oligonucleotides complementary to oneof the COS ends and attached to carboxylated surfaces, as describedabove. The hybridization conditions (pure water at 40° C.) were notstringent, because under stringent conditions (high salinity) thefluorescence of the YOYO1 probes disappears and the hybridized DNAscannot be seen. It was also possible to align the DNAs thus hybridizedby passage of the meniscus.

Sensitivity of the Detection

In order to determine the sensitivity of the detection method byextension of the meniscus, 2.5 μl drops of a solution of λ DNA in 0.05 MMES (pH=5.5) containing a total of 10⁵, 10⁴ and 1000 molecules, weredeposited on double bond surfaces. The anchoring and the alignment arecarried out as described above. The cover slips are then observed byepifluorescence microscopy to determine the density of combed molecules.The latter indeed corresponds to that estimated: about 4-6 DNA moleculesper field of vision (100 μm×100 μm) for a total of 10⁵ DNA molecules.For the lowest concentration, it was possible to observe about tenmolecules extended by the action of the meniscus. This number isessentially limited by the large number of fields of vision required tocover the whole sample (about 25,000), which makes a manual searchdifficult, but it can be advantageously carried out automatically oralso with a weaker lens, but with a larger field. In conclusion, thesensitivity of the method according to the invention allows detectionand individual counting of less than 1000 DNA molecules.

Dependence of the Stretching on the Surface Treatment

The histogram of the lengths of λ DNA grafted on different surfaces andthen aligned by passage of the meniscus shows a well defined peak butwhich is different for the different surfaces. Thus, on surfaces coatedwith a silane which end with a vinyl group, the DNA is stretched up toabout 22 μm (see above) for surfaces silanized with an amine group(—NH₂), the histogram has a peak at 21 μm (FIG. 7(a)) and on clean glassat about 18.5 μm (FIG. 7(c)).

The stretching therefore depends on the surface treatment.

EXAMPLE 2

Combing of DNA Molecules on Different Surfaces

The molecular combing of DNA on glass surfaces treated in various wayswas observed. Advantage is taken of the difference in adsorption betweenthe ends of the molecule and the rest of the molecule. By adsorbingpositively charged polyners onto a glass surface, adsorption ofnegatively charged DNA molecules is enhanced, however when this chargeis large, the DNA molecule is stuck over its entire length and thecombing is impossible. However it is possible to modify the charge ofthe polymers adsorbed on the glass by modifying the pH conditions,indeed, the positive charges are carried for example by the NN2 groupswhich pass to the protonated state NE₃+ for a pH below the pK of thecorresponding base. In basic pH, the charges disappear and the surfaceno longer attracts DNA. By finely controlling the pH, it was observedthat the DNA molecules in solution passed from a state where they arecompletely stuck to the surface to an intermediate phase where they areanchored only by their ends and then to a phase where the surface nolonger has affinity for the DNA. In the intermediate phase, molecularcombing can be carried out.

Surfaces coated with a silane ending with an NH₂ group were studied forwhich there is observed complete sticking at pH<8, and combing for8.5<pH<9.5. The number of combed molecules is maximum at pH=8.5 it isdivided by 2 at pH=9 and by 4 at pH=9.5. Also the relative extension onthis surface which corresponds to 1.26 was determined as can be seen inhistogram 2 of FIG. 7 which represents histograms of the length of thecombed λ DNA molecules on glass surfaces:

-   -   a) coated with silane ending with an amine group,    -   b) coated with polylysine,    -   c) cleaned in a hydrogen peroxide/sulfuric acid mixture.

Surfaces coated with polylysine were also examined and found to exhibitsimilar attachment characteristics as regards the pH: combing region at8.5 and exhibiting a shorter relative extension: 1.08. A typical examplecan be obtained in FIG. 8 which represents combed DNA molecules on glasssurfaces coated with polylysine. It can be observed that the moleculesattached by their two ends form loops.

Finally, the same behavior was found on glass surfaces freshly cleanedin a hydrogen peroxide/concentrated sulfuric acid mixture. Thesesurfaces are highly wetting and become rapidly contaminated; however, acombing region was observed between 5.5<pH<7.4 whereas the region ofstrong adsorption is situated at pH=4.5. The relative extension of themolecules corresponds to 1.12.

EXAMPLE 3

Uniform and Directional Alignment of YAC

1 μg of YAC previously stained in its agarose plug by means of a YOYO1fluorescent probe is heated to 68° C., agarased and then diluted in 10ml of MES (50 mM pH 5.5). Two silanized cover slips (C═C surfaces) areincubated for ≈1.5 h in this solution and then removed at about 170μm/sec. The YAC molecules are all aligned parallel to the direction ofremoval of the cover slips (FIG. 9). The integrity of the molecules thusaligned is better than by evaporation after deposition between two coverslips.

Hybridization of a Cosmid with a Combed YAC

A YAC stained as previously described is anchored on a C═C surface(between two cover slips) and then aligned by the meniscus, duringevaporation of the solution. The probes (cosmids) are labeled byincorporation of a biotinylated nucleotide by the random primingtechnique. The labeled probes (100 ng) and 5 μg of sonicated salmonsperm DNA (≈500 bps) are purified by precipitation in Na-acetate andethanol, and then denatured in formamide.

The combed YACs are denatured between two cover slips with 120 μl ofdenaturing solution (70% formamide, 2×SSC) on a hotplate at 80° C. for 3minutes. The previously denatured probes (20 ng) are deposited on thecover slip in a hybridization solution (55% formamide, 2×SSC, 10%dextran sulfate) covered with a cover slip and sealed with rubbercement. The hybridization is carried out overnight at 37° C. in a humidchamber.

The detection of the hybrids is performed according to procedures knownfor in situ hybridizations on decondensed chromosomes (D. Pinkel et al.,PNAS USA 83, 2934 (1986) and PNAS USA 85, 9138 (1988)).

Hybridized segments such as that shown in FIG. 10 are then observed byfluorescence microscopy. This example demonstrates the possibility ofdetecting the presence of a gene on a DNA molecule, which can be usedfor diagnostic purposes or for physical mapping of the genome.

1-49. (canceled)
 50. A process for aligning a macromolecule on asurface, wherein the process comprises: (a) providing a solvent for themacromolecule between two surfaces where one surface is an anchoringsurface to which one end of the macromolecule is anchored by a bond andanother end of the macromolecule is free, and where the solvent forms ameniscus between and perpendicular to the surfaces at an interface ofthe solvent with air between the surfaces; and (b) moving the meniscusby gradual evaporation of the solvent so that the macromolecule passesthrough the meniscus into air at the interface whereby the macromoleculeis aligned on the anchoring surface perpendicular to the meniscuswithout breaking the bond that anchors the end of the macromolecule tothe surface.
 51. A process according to claim 50, wherein themacromolecule is a protein, a lipid, a polysaccharide, or a derivativethereof.
 52. A process according to claim 50, wherein the macromoleculeis an antibody, an antigen, a ligand, a receptor of a ligand, or aderivative thereof.
 53. A process according to claim 50, wherein themacromolecule is a protein capable of specifically recognizing andattaching a second protein in a sample.
 54. A process according to claim50, wherein the macromolecule is a protein.
 55. A process according toclaim 50, wherein the anchoring surface comprises (a) an exposedreactive group having an affinity for the macromolecule, or (b) amolecule with biological activity capable of recognizing themacromolecule.
 56. A process according to claim 55, wherein the moleculewith biological activity is biotin, avidin, streptavidin, or derivativesthereof, an antigen, or an antibody.